Assay devices that utilize hollow particles

ABSTRACT

Hollow particles for use in various types of assay devices are provided. Due to their hollow or voided structure, the particles may exhibit a variety of beneficial properties. For instance, hollow particles are generally lightweight, and thus, relatively inexpensive in comparison to other types of particles. Hollow particles may also form a stable system without requiring refrigeration or rotation. In addition, hollow particles may possess enhanced light diffraction capabilities, which may be particularly beneficial in certain types of assay devices, e.g., diffraction-based assay devices.

BACKGROUND OF THE INVENTION

[0001] Various analytical procedures and devices are commonly employedto determine the presence and/or concentration of analytes that may bepresent in a test sample. For instance, immunoassays utilize mechanismsof the immune systems, wherein antibodies are produced in response tothe presence of antigens that are pathogenic or foreign to theorganisms. These antibodies and antigens, i.e., immunoreactants, arecapable of binding with one another, thereby causing a highly specificreaction mechanism that can be used to determine the presence orconcentration of that particular antigen in a biological sample.

[0002] In many assay devices, labeled particles are used to signal thepresence or absence of the analyte of interest, either visually orthough the use of an instrument. For instance, commercial examples offluorescent carboxylated microspheres are available from MolecularProbes, Inc. under the trade names “FluoSphere” (Red 580/605) and“TransfluoSphere” (543/620). Commercial examples of colored carboxylatedlatex beads are also available from Bang's Laboratory, Inc. Goldparticles are also commonly utilized.

[0003] Despite some success, conventional particles still possessnumerous problems when used in assay devices. For instance, conventionallatex beads tend to aggregate with each other, thus requiring that theybe refrigerated and kept under continuous agitation until use. Thisaggregation may lead to poor reliability and reproducibility in theassay device. Further, although gold particles have a relatively lowersize distribution and do not tend to aggregate as much as latex beads,they are difficult and expensive to manufacture. Many of thecommercially available gold particles are also poor in quality.Moreover, most gold particles have a red color that may not be changed,leading to less flexibility in the assay format and design.

[0004] As such, a need currently exists for improved particles forincorporation into an assay device.

SUMMARY OF THE INVENTION

[0005] In accordance with one embodiment of the present invention, anassay device (e.g., flow-through assay device, diffraction-based assaydevice, etc.) is disclosed that comprises a plurality of detectableprobes, wherein at least one of the detectable probes contains aparticle that defines a hollow interior constituting from about 20% toabout 100% of the spatial volume occupied by the particle.

[0006] The shape of the particle may generally vary as desired. Forexample, in some embodiments, the particle may have the shape of asphere, plate, rod, disc, bar, tube, an irregular shape, etc. Inaddition, the size of the particle may also vary. For example, in someembodiments, the average size of the particle may range from about 0.1nanometers to about 1,000 microns, in some embodiments from about 0.1nanometers to about 100 microns, and in some embodiments, from about 1nanometer to about 10 microns.

[0007] Besides size and/or shape, the material(s) that form the hollowparticle may also vary. The hollow particle may, for instance, beorganic and/or inorganic in nature, and may be polymers, oligomers,molecules, and so forth. For instance, in one embodiment, the particleis formed from a core polymer and a shell polymer. In anotherembodiment, the particle is formed by electrostatic layer deposition.The particle may be modified in any manner desired to facilitate its usein the assay device. For example, in some embodiments, the particle isconjugated with a specific binding member. In one embodiment, thespecific binding member is covalently bonded to the particle.

[0008] In accordance with another embodiment of the present invention, adetectable probe for use in an assay device is disclosed. The detectableprobe contains a particle that defines a hollow interior constitutingfrom about 20% to about 100% of the spatial volume occupied by theparticle.

[0009] Other features and aspects of the present invention are discussedin greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] A full and enabling disclosure of the present invention,including the best mode thereof, directed to one of ordinary skill inthe art, is set forth more particularly in the remainder of thespecification, which makes reference to the appended figures in which:

[0011]FIG. 1 is a perspective view of one embodiment of a flow-throughassay device of the present invention;

[0012]FIG. 2 is a perspective view of one embodiment of adiffraction-based assay device of the present invention;

[0013]FIG. 3 is a graphical illustration of one embodiment forcovalently conjugating an antibody to carboxylated hollow particles; and

[0014]FIG. 4 is an SEM photograph (magnification of 100×) of the hollowparticles utilized in Example 1.

[0015] Repeat use of reference characters in the present specificationand drawings is intended to represent same or analogous features orelements of the invention.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS Definitions

[0016] As used herein, the term “analyte” generally refers to asubstance to be detected. For instance, analytes can include antigenicsubstances, haptens, antibodies, and combinations thereof. Analytesinclude, but are not limited to, toxins, organic compounds, proteins,peptides, microorganisms, amino acids, nucleic acids, hormones,steroids, vitamins, drugs (including those administered for therapeuticpurposes as well as those administered for illicit purposes), drugintermediaries or byproducts, bacteria, virus particles and metabolitesof or antibodies to any of the above substances. Specific examples ofsome analytes include ferritin; creatinine kinase MIB (CK-MB); digoxin;phenytoin; phenobarbitol; carbamazepine; vancomycin; gentamycin;theophylline; valproic acid; quinidine; leutinizing hormone (LH);follicle stimulating hormone (FSH); estradiol, progesterone; C-reactiveprotein; lipocalins; IgE antibodies; vitamin B2 micro-globulin; glycatedhemoglobin (Gly. Hb); cortisol; digitoxin; N-acetylprocainamide (NAPA);procainamide; antibodies to rubella, such as rubella-IgG and rubellaIgM; antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG)and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylates;acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies tohepatitis B core antigen, such as anti-hepatitis B core antigen IgG andIgM (Anti-HBC); human immune deficiency virus 1 and 2 (HIV 1 and 2);human T-cell leukemia virus 1 and 2 (HTLV); hepatitis B e antigen(HBeAg); antibodies to hepatitis B e antigen (Anti-HBe); influenzavirus; thyroid stimulating hormone (TSH); thyroxine (T4); totaltriiodothyronine (Total T3); free triiodothyronine (Free T3);carcinoembryoic antigen (CEA); and alpha fetal protein (AFP). Drugs ofabuse and controlled substances include, but are not intended to belimited to, amphetamine; methamphetamine; barbiturates, such asamobarbital, secobarbital, pentobarbital, phenobarbital, and barbital;benzodiazepines, such as librium and valium; cannabinoids, such ashashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates,such as heroin, morphine, codeine, hydromorphone, hydrocodone,methadone, oxycodone, oxymorphone and opium; phencyclidine; andpropoxyhene. Other potential analytes may be described in U.S. Pat. No.6,436,651 to Everhart, et al. and U.S. Pat. No. 4,366,241 to Tom et al.

[0017] As used herein, the term “test sample” generally refers to amaterial suspected of containing the analyte. The test sample can beused directly as obtained from the source or following a pretreatment tomodify the character of the sample. The test sample can be derived fromany biological source, such as a physiological fluid, including, blood,interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid,sweat, urine, milk, ascites fluid, raucous, synovial fluid, peritonealfluid, vaginal fluid, amniotic fluid or the like. The test sample can bepretreated prior to use, such as preparing plasma from blood, dilutingviscous fluids, and the like. Methods of treatment can involvefiltration, precipitation, dilution, distillation, mixing,concentration, inactivation of interfering components, and the additionof reagents. Besides physiological fluids, other liquid samples can beused such as water, food products and the like for the performance ofenvironmental or food production assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample. Insome instances it may be beneficial to modify a solid test sample toform a liquid medium or to release the analyte.

DETAILED DESCRIPTION

[0018] Reference now will be made in detail to various embodiments ofthe invention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment, can be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncovers such modifications and variations as come within the scope of theappended claims and their equivalents.

[0019] The present invention is generally directed to the use of hollowparticles in various types of assay devices. Generally speaking, thehollow particles individually define a hollow interior that constitutesfrom about 20% to about 100%, and in some embodiments, from about 30% toabout 100% of the spatial volume occupied by the particle. Namely, asubstantial portion of the spatial volume of each hollow particleremains empty. Due to their hollow or voided structure, the particlesmay exhibit a variety of beneficial properties. For instance, hollowparticles are generally lightweight, and thus, sometimes inexpensive incomparison to other types of particles. Hollow particles, may also forma stable system without requiring refrigeration or rotation, and may bereadily resuspended when it is desired to use the particles. Inaddition, hollow particles may possess enhanced light diffractioncapabilities, which may be particularly beneficial in certain types ofassay devices, e.g., diffraction-based assay devices.

[0020] The shape of the hollow particles may generally vary. In oneparticular embodiment, for instance, the hollow particles are sphericalin shape. However, it should be understood that other shapes are alsocontemplated by the present invention, such as plates, rods, discs,bars, tubes, irregular shapes, etc.

[0021] In addition, the size of the hollow particles may also vary. Forinstance, the average size (e.g., diameter) of the hollow particles mayrange from about 0.1 nanometers to about 1,000 microns, in someembodiments, from about 0.1 nanometers to about 100 microns, and in someembodiments, from about 1 nanometer to about 10 microns. The sizeselected for the hollow microparticles may depend on the intendedapplication. For instance, “micron-scale” particles may be desired insome assay devices, such as some flow-through devices ordiffraction-based assays. When utilized, such “micron-scale” particlesmay have an average size of from about 1 micron to about 1,000 microns,in some embodiments from about 1 micron to about 100 microns, and insome embodiments, from about 1 micron to about 10 microns. Likewise,“nano-scale” particles may be desired in other applications, such as insome flow-through assay devices. When utilized, such “nano-scale”particles may have an average size of from about 0.1 to about 10nanometers, in some embodiments from about 0.1 to about 5 nanometers,and in some embodiments, from about 1 to about 5 nanometers.

[0022] Although the shape and size of the particles may vary, asdescribed above, it is often desired that the particles may berelatively “monodispersed” in that the particles within a givendispersion have approximately the same size and/or shape. Monodispersedhollow particles can provide improved reliability and reproducibilitydue to their generally uniform properties.

[0023] Besides their size and shape, the material(s) that form thehollow particles may also vary. The hollow particles may, for instance,be organic and/or inorganic in nature, and may be polymers, oligomers,molecules, and so forth. For example, the hollow particles may be formedfrom polymers such as polystyrene, (meth)acrylate polymers orcopolymers, vinylidene chloride/acrylonitrile copolymers, etc. Othersuitable hollow polymeric particles may be described in U.S. Pat. No.4,427,836 to Kowalski, et al.; U.S. Pat. No. 4,480,042 to Craig, et al.;U.S. Pat. No. 4,973,670 to McDonald, et al.; U.S. Pat. No. 5,618,888 toChoi, et al.; and U.S. Pat. No. 6,139,961 to Blankenship, et al., whichare incorporated herein in their entirety by reference thereto for allpurposes. Still other hollow particles that may be used includeinorganic materials, such as glass hollow particles. For instance,ECCOSPHERES® are hollow glass particles derived from sodium borosilicatecommercially available from Emerson and Cuming Composite Materials, Inc.Other representative hollow particles derived from an inorganicmaterial, include, for instance, silica hollow microspheres availableunder the trade name “SILICA BEADS S700” from Miyoshi Kasei, Inc. Otherexamples of hollow inorganic particles are described in U.S. Pat. No.6,416,774 to Radin, et al., which is incorporated herein in its entiretyby reference thereto for all purposes.

[0024] In one particular, embodiment, the hollow particles may be formedfrom one or more natural or synthetic latex polymers. Examples of suchlatex-based hollow particles are described in U.S. Pat. No. 5,663,213 toJones, et al., which is incorporated herein in its entirety by referencethereto for all purposes, and commercially available from Rohm & Haas ofPhiladelphia, Pa. under the name SunSpheres®. The '213 patent describesthe ability of such latex-based hollow particles, which are typically“micron-scale” in size, to be used for sun protection. However, thepresent inventors have also discovered that the latex-based hollowparticles have unexpected utility in assay devices.

[0025] The latex-based hollow particles typically contain a core polymerand a shell polymer. The monomers used to form the core and shellpolymers may generally vary. For instance, the shell polymer may beselected to provide a glass transition temperature (T_(g)) that is highenough to support the voids of the particle, e.g., such as greater thanabout 50° C., in some embodiments greater than about 60° C., and in someembodiments, greater than about 70° C. Some examples of suitablemonomers that may be used to form the shell polymer include, but are notlimited to, non-ionic ethylenically unsaturated monomers,monoethylenically unsaturated monomers containing at least onecarboxylic acid group, and so forth.

[0026] The monomers that form the core polymer may include one or moremonoethylenically unsaturated monomers containing at least onecarboxylic acid group. In some embodiments, for instance, at least about5 wt. % of the monoethylenically unsaturated monomers of the corepolymer contain at least one carboxylic acid, based on total monomerweight of the core. Examples of suitable monoethylenically unsaturatedmonomers containing at least one carboxylic acid group include, but arenot limited to, (meth)acrylic acid, acryloxypropionic acid,(meth)acryloxypropionic acid, itaconic acid, aconitic acid, maleic acidor anhydride, fumaric acid, crotonic acid, monomethyl maleate,monomethyl fumarate, monomethyl itaconate, and so forth. As used herein,the term “(meth)acrylic” is intended to serve as a generic expressionembracing both acrylic and methacrylic.

[0027] In one embodiment, the monoethylenically unsaturated monomercontaining at least one carboxylic acid group is copolymerized with oneor more nonionic (e.g., having no ionizable group) ethylenicallyunsaturated monomers. Some suitable nonionic ethylenically unsaturatedmonomers include, but are not limited to, styrene, vinyltoluene,ethylene, vinyl acetate, vinyl chloride, vinylidene chloride,acrylonitrile, (meth)acrylamide, (C₁-C₂₀) alkyl or (C₃-C₂₀) alkenylesters of (meth)acrylic acid, such as methyl (meth)acrylate, ethyl(meth)acrylate, butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate,benzyl (meth)acrylate, lauryl (meth)acrylate, oleyl (meth)acrylate,palmityl (meth)acrylate, stearyl (meth)acrylate, and so forth.

[0028] The core polymer and/or shell polymer may optionally contain fromabout 0.1 wt. % to about 20 wt. %, and in some embodiments, from about0.1 wt. % to about 3 wt. % of a polyethylenically unsaturated monomerbased on the total monomer weight of the polymer. Examples of suchunsaturated monomers include, but are not limited to, ethylene glycoldi(meth)acrylate, allyl(meth)acrylate, 1,3-butanediol di(meth)acrylate,diethylene glycol di(meth)acrylate, trimethylolpropanetri(meth)acrylate, or divinylbenzene. If desired, the core polymerand/or shell polymer may contain from about 0.1 wt. % to about 60 wt. %butadiene based on the total monomer weight of the polymer.

[0029] To produce the void in the latex particles, the core is typicallyswelled with a swelling agent containing one or more volatilecomponents. The swelling agent permeates the shell to swell the core.The volatile components of the swelling agent may then be removed bydrying the latex particles, thereby causing a void to form within thelatex particles. Although not required, the swelling agent may be anaqueous base. Examples of suitable aqueous bases include, but are notlimited to, ammonia, ammonium hydroxide, alkali metal hydroxides, suchas sodium hydroxide, or a volatile amine, such as trimethylamine ortriethylamine. Removal of the templated core may also be accomplished inother ways, such as by calcining at elevated temperatures or by chemicalreactions causing dissolution of the core material.

[0030] In addition to core-shell hollow particles, hollow particles mayalso be formed using other well-known techniques. For example, U.S. Pat.No. 6,479,146 to Caruso, et al., which is incorporated herein in itsentirety by reference thereto for all purposes, describes hollowparticles formed using electrostatic forces. In particular, hollowparticles are formed using colloid templated electrostaticlayer-by-layer (“LBL”) self-assembly of nanoparticle-polymermultilayers, followed by removal of the templated core. The templateparticles may, for instance, contain organic polymer latices, such aspolystyrene or styrene copolymer latices.

[0031] The template particles are alternately coated withpolyelectrolyte molecules and nanoparticles. The polyelectrolytes areusually polymers having ionically dissociable groups that may be acomponent or substituent of the polymer chain. The nanoparticles aretypically ceramic particles, such as silicon dioxide, titanium dioxide,and zirconium dioxide optionally doped with other metal oxides; magneticparticles, such as Fe₃O₄; magneto-optical particles; nitridic ceramicparticles, such as Si₃N₄, carbidic ceramic particles; metallicparticles, such as gold, silver, and palladium; and sulfur orselene-containing particles, such as cadmium sulfide, cadmium selenideetc.

[0032] In one embodiment, the template particles are first coated withseveral layers of oppositely charged cationic and anionicpolyelectrolytes before the alternating layers of nanoparticles andpolyelectrolyte or the alternating nanoparticle layers are applied. Thetemplate particles may be coated with at least two and up to six layersof oppositely charged cationic and anionic polyelectrolytes, e.g., withthree layers. The outermost polyelectrolyte layer may be oppositelycharged with regard to the nanoparticle to be deposited. In mostembodiments, the template particles are at least partially disintegratedafter the coating has been completed. They can be dissolved inappropriate solvents or thermally (e.g., by calcination to temperaturesof at least about 500° C.). After dissolution of the template particles,hollow shells remain that are composed of the nanoparticle material andoptionally the polyelectrolyte material.

[0033] If desired, the electrostatically-formed particles may bemodified to contain pores in at least one of the layers. Such pores canbe formed by the polyelectrolytes or nanoparticles themselves. Forinstance, a high salt concentration of the medium used for thedeposition of the polyelectrolyte may result in a high permeability ofthe shell wall. On the other hand, a high salt concentration of themedium used for the deposition of the nanoparticles (e.g., SiO₂) mayresults in a high packing density of the silica particles. Thus, byadjusting the salt concentrations in the deposition medium, thepermeability of the shell can be controlled, as desired. Further, thepermeability properties of the shell may be modified by selecting theconditions for decomposing the core, e.g., by selecting the temperatureand heating conditions in a calcination procedure.

[0034] Hollow particles, such as described above, may have a variety ofuses in assay devices. For instance, in one particular embodiment, thehollow particles may be used as probes in an assay device. When utilizedas probes, the hollow particles may be capable of inherently generatinga signal that is detectable visually or by an instrumental device (e.g.,a diffraction pattern), or may be modified with a label to impart such asignal generation capability. Various suitable labels that can be usedinclude, but are not limited to, chromogones; catalysts; fluorescentcompounds; chemiluminescent compounds; phosphorescent compounds;radioactive compounds; direct visual labels; and the like. For instance,some suitable labels may be described in U.S. Pat. No. 5,670,381 to Jou,et al. and U.S. Pat. No. 5,252,459 to Tarcha, et al., which areincorporated herein in their entirety by reference thereto for allpurposes. In one particular embodiment, the label is a fluorescentcompound that produces a detectable signal. The fluorescent compoundscan be fluorescent molecules, polymers, dendrimers, particles, and thelike. Some examples of suitable fluorescent molecules, for instance,include, but are not limited to, fluorescein, europium chelates,phycobiliprotein, rhodamine and their derivatives and analogs. Avisually detectable, colored compound can also be used as a label,thereby providing for a direct colored readout of the presence orconcentration of the analyte in the sample without the need for furthersignal producing reagents.

[0035] The hollow particles may also be modified in some manner so thatthey are more readily able to bond with an analyte. In such instances,the hollow particles can be modified with certain specific bindingmembers that are adhered thereto to form conjugated probes. Specificbinding members generally refer to a member of a specific binding pair,i.e., two different molecules where one of the molecules chemicallyand/or physically binds to the second molecule. For instance,immunoreactive specific binding members can include antigens, haptens,aptamers, antibodies, and complexes thereof, including those formed byrecombinant DNA methods or peptide synthesis. An antibody can be amonoclonal or polyclonal antibody, a recombinant protein or a mixture(s)or fragment(s) thereof, as well as a mixture of an antibody and otherspecific binding members. The details of the preparation of suchantibodies and their suitability for use as specific binding members arewell known to those skilled in the art. Other common specific bindingpairs include but are not limited to, biotin and avidin, biotin andstreptavidin, antibody-binding proteins (such as protein A or G) andantibodies, carbohydrates and lectins, complementary nucleotidesequences (including label and capture nucleic acid sequences used inDNA hybridization assays to detect a target nucleic acid sequence),complementary peptide sequences including those formed by recombinantmethods, effector and receptor molecules, hormone and hormone bindingprotein, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,and the like. Furthermore, specific binding pairs can include membersthat are analogs of the original specific binding member. For example, aderivative or fragment of the analyte, i.e., an analyte-analog, can beused so long as it has at least one epitope in common with the analyte.

[0036] The specific binding members can generally be attached to thehollow particles using any of a variety of well-known techniques. Forinstance, covalent attachment of the specific binding members to thehollow particles can be accomplished using carboxylic, amino, aldehyde,bromoacetyl, iodoacetyl, thiol, epoxy and other reactive or linkingfunctional groups, as well as residual free radicals and radicalcations, through which a protein coupling reaction can be accomplished.A surface functional group can also be incorporated as a functionalizedco-monomer because the surface of the hollow particles can contain arelatively high surface concentration of polar groups. In addition,although the hollow probes are often functionalized after synthesis, incertain cases, the hollow particles may be capable of direct covalentlinking with a protein without the need for further modification. Forexample, referring to FIG. 3, one embodiment of the present inventionfor covalently conjugating a hollow particle is illustrated. In thisembodiment, the hollow particles contain carboxylic functional groups,which may be present on latex-based hollow particles, such as describedabove. As shown, the first step of conjugation is activation ofcarboxylic groups on the hollow particle surface using carbodiimide. Inthe second step, the activated carboxylic acid groups are reacted withan amino group of an antibody to form an amide bond. The activationand/or antibody coupling can occur in a buffer, such asphosphate-buffered saline (PBS) (e.g., pH of 7.2) or 2-(N-morpholino)ethane sulfonic acid (MES) (e.g., pH of 5.3). As shown, the resultinghollow particles can then be blocked with ethanolamine, for instance, toblock any remaining activated sites. Overall, this process forms aconjugate, where the antibody is covalently attached to the particle.Besides covalent bonding, other attachment techniques, such as physicaladsorption, may also be utilized in the present invention.

[0037] Hollow particles, such as described above, may be incorporatedinto a variety of assay devices. Examples of some suitable assay devicesthat may employ the hollow particles include, but are not limited to,flow through-assay devices (membrane-based, fluidic-based,capillary-based, etc.); diffraction-based assay devices; and so forth.Specifically, such assay devices often utilize probes that aredetectable in some manner, such as visually or through the use of aninstrument. Such detectable probes may be used for detecting the analyteand/or for calibration of the assay device. In accordance with thepresent invention, the hollow particles may be utilized as detectableprobes for purposes of detection and/or calibration. Of course, itshould also be understood that the hollow particles may be used in anassay device according to the present invention in other ways notspecifically referenced herein.

[0038] For purposes of illustration only, various examples of assaydevices that may incorporate hollow particles according to the presentinvention will now be described in more detail. It should be understood,however, that other assay devices are also contemplated by the presentinvention. In fact, the present invention is not limited to anyparticular assay device configuration.

[0039] Referring to FIG. 1, for instance, one embodiment of amembrane-based flow-through assay device 20 is illustrated. As shown,the device 20 contains a porous membrane 23 optionally supported by arigid material 21. In general, the porous membrane 23 can be made fromany of a variety of materials through which the test sample is capableof passing. For example, the materials used to form the porous membrane23 can include, but are not limited to, natural, synthetic, or naturallyoccurring materials that are synthetically modified, such aspolysaccharides (e.g., cellulose materials such as paper and cellulosederivatives, such as cellulose acetate and nitrocellulose); silica;inorganic materials, such as deactivated alumina, diatomaceous earth,MgSO₄, or other inorganic finely divided material uniformly dispersed ina porous polymer matrix, with polymers such as vinyl chloride, vinylchloride-propylene copolymer, and vinyl chloride-vinyl acetatecopolymer; cloth, both naturally occurring (e.g., cotton) and synthetic(e.g., nylon or rayon); porous gels, such as silica gel, agarose,dextran, and gelatin; polymeric films, such as polyacrylamide; and thelike. In one particular embodiment, the porous membrane 23 is formedfrom nitrocellulose and/or polyester sulfone materials. It should beunderstood that the term “nitrocellulose” refers to nitric acid estersof cellulose, which may be nitrocellulose alone, or a mixed ester ofnitric acid and other acids, such as aliphatic carboxylic acids havingfrom 1 to 7 carbon atoms.

[0040] The device 20 may also contain a wicking pad 28. The wicking pad28 generally receives fluid that has migrated through the entire porousmembrane 23. As is well known in the art, the wicking pad 28 can assistin promoting capillary action and fluid flow through the membrane 23.

[0041] To initiate the detection of an analyte within the test sample, auser may directly apply the test sample to a portion of the porousmembrane 23 through which it can then travel. Alternatively, the testsample may first be applied to a sampling pad (not shown) that is influid communication with the porous membrane 23. Some suitable materialsthat can be used to form the sampling pad include, but are not limitedto, nitrocellulose, cellulose, porous polyethylene pads, and glass fiberfilter paper. If desired, the sampling pad may also contain one or moreassay pretreatment reagents, either diffusively or non-diffusivelyattached thereto.

[0042] In the illustrated embodiment, the test sample travels from thesampling pad (not shown) to a conjugate pad 22 that is placed incommunication with one end of the sampling pad. The conjugate pad 22 isformed from a material through which the test sample is capable ofpassing. For example, in one embodiment, the conjugate pad 22 is formedfrom glass fibers. Although only one conjugate pad 22 is shown, itshould be understood that other conjugate pads may also be used in thepresent invention.

[0043] To facilitate detection of the presence or absence of an analytewithin the test sample, various detection probes 41, which optionallycontain the hollow particles of the present invention, may be applied tothe conjugate pad 22. While contained on the conjugate pad 22, thesedetection probes 41 remain available for binding with the analyte as itpasses from the sampling pad through the conjugate pad 22. Upon bindingwith the analyte, the detection probes 41 can later serve to identifythe presence or absence of the analyte. The detection probes 41 may beused for both detection and calibration of the device 20. In alternativeembodiments, however, separate calibration probes 43, which alsooptionally contain the hollow particles of the present invention, may beapplied to the conjugate pad 22 for use in conjunction with thedetection probes 41 to facilitate simultaneous calibration anddetection, thereby eliminating inaccuracies often created byconventional assay calibration systems. It should be understood,however, that the detection probes 41 and/or the calibration probes 43may be applied together or separately at any location of the device 20,and need not be applied to the conjugate pad 22. Further, it should alsobe understood that the detection probes 41 and/or the calibration probes43 may be applied to the same or different conjugate pads.

[0044] In one embodiment, for instance, the test sample travels to theconjugate pad 22, where the analyte mixes with the detection probes 41to form analyte complexes. Because the conjugate pad 22 is in fluidcommunication with the porous membrane 23, the complexes can migratefrom the conjugate pad 22 to a detection zone 31 present on the porousmembrane 23. The detection zone 31 may contain an immobilized receptivematerial that is generally capable of forming a chemical or physicalbond with the probes. For example, in some embodiments, the binders cancontain a biological receptive material. For example, in someembodiments, the receptive material may be a biological receptivematerial. Such biological receptive materials are well known in the artand can include, but are not limited to, antigens, haptens, antibodies,protein A or G, avidin, streptavidin, secondary antibodies, andcomplexes thereof. In some cases, it is desired that these biologicalreceptive materials are capable of binding to a specific binding member(e.g., antibody) present on the hollow particles. In addition, it mayalso be desired to utilize various non-biological materials for thereceptive material. For instance, in some embodiments, the receptivematerial can include a polyelectrolyte that can bind to the uncapturedprobes. The polyelectrolytes can have a net positive or negative charge,as well as a net charge that is generally neutral. For instance, somesuitable examples of polyelectrolytes having a net positive chargeinclude, but are not limited to, polylysine (commercially available fromSigma-Aldrich Chemical Co., Inc. of St. Louis, Mo.), polyethylenimine;epichlorohydrin-functionalized polyamines and/or polyamidoamines, suchas poly(dimethylamine-co-epichlorohydrin); polydiallyidimethyl-ammoniumchloride; cationic cellulose derivatives, such as cellulose copolymersor cellulose derivatives grafted with a quaternary ammoniumwater-soluble monomer; and the like. In one particular embodiment,CelQuat® SC-230M or H-100 (available from National Starch & Chemical,Inc.), which are cellulosic derivatives containing a quaternary ammoniumwater-soluble monomer, can be utilized. Moreover, some suitable examplesof polyelectrolytes having a net negative charge include, but are notlimited to, polyacrylic acids, such as poly(ethylene-co-methacrylicacid, sodium salt), and the like. It should also be understood thatother polyelectrolytes may also be utilized in the present invention,such as amphiphilic polyelectrolytes (i.e., having polar and non-polarportions). For instance, some examples of suitable amphiphilicpolyelectrolytes include, but are not limited to, poly(styryl-b-N-methyl2-vinyl pyridinium iodide) and poly(styryl-b-acrylic acid), both ofwhich are available from Polymer Source, Inc. of Dorval, Canada.

[0045] These receptive materials serve as stationary binding sites forthe detection probe/analyte complexes. In some instances, the analytes,such as antibodies, antigens, etc., have two binding sites. Uponreaching the detection zone 31, one of these binding sites is occupiedby the specific binding member of the complexed probes. However, thefree binding site of the analyte can bind to the immobilized receptivematerial. Upon being bound to the immobilized receptive material, thecomplexed probes form a new ternary sandwich complex.

[0046] The detection zone 31 may generally provide any number ofdistinct detection regions so that a user can better determine theconcentration of a particular analyte within a test sample. Each regionmay contain the same receptive materials, or may contain differentreceptive materials for capturing multiple analytes. For example, thedetection zone 31 may include two or more distinct detection regions(e.g., lines, dots, etc.). The detection regions may be disposed in theform of lines in a direction that is substantially perpendicular to theflow of the test sample through the assay device 20. Likewise, in someembodiments, the detection regions can be disposed in the form of linesin a direction that is substantially parallel to the flow of the testsample through the assay device.

[0047] Although the detection zone 31 may indicate the presence of ananalyte, it is often difficult to determine the relative concentrationof the analyte within the test sample using solely a detection zone 31.Thus, the assay device 20 may also include a calibration zone 32. Inthis embodiment, the calibration zone 32 is formed on the porousmembrane 23 and is positioned downstream from the detection zone 31. Thecalibration zone 32 is provided with a receptive material that iscapable of binding to any remaining uncaptured detection probes 41and/or calibration probes 43 that pass through the length of themembrane 23. In particular, upon being contacted with the test sample,any uncaptured probes that do not bind to the analyte migrate throughthe detection zone 31 and enter the calibration zone 32 of the porousmembrane 23. At the calibration zone 32, these uncaptured probes thenbind to the receptive materials. The receptive materials utilized in thecalibration zone 32 may be the same or different than the receptivematerials used in the detection zone 31. Moreover, similar to thedetection zone 31, the calibration zone 32 may also provide any numberof distinct calibration regions in any direction so that a user canbetter determine the concentration of a particular analyte within a testsample. Each region may contain the same receptive materials, or maycontain different receptive materials for capturing different probes.

[0048] The calibration regions may be pre-loaded on the porous membrane23 with different amounts of the binder so that a different signalintensity is generated by each calibration region upon migration of theuncaptured probes. The overall amount of receptive material within eachcalibration region can be varied by utilizing calibration regions ofdifferent sizes and/or by varying the concentration or volume of thebinder in each calibration region. If desired, an excess of probemolecules can be employed in the assay device 20 so that eachcalibration region reaches its full and predetermined potential forsignal intensity. That is, the amount of uncaptured probes that aredeposited upon calibration regions are predetermined because the amountof the binder employed on the calibration regions is set at apredetermined and known level. Once captured, the signal of the probesat the detection and calibration zones 31 and 32 can be measured usingvisually and/or with an instrument.

[0049] Various formats may be used to test for the presence or absenceof an analyte using the device 20. For instance, in the embodimentdescribed above, a “sandwich” format is utilized. Other examples of suchsandwich-type assays are described by U.S. Pat. No. 4,168,146 to Grubb,et al. and U.S. Pat. No. 4,366,241 to Tom, et al., which areincorporated herein in their entirety by reference thereto for allpurposes. In addition, other formats, such as “competitive” formats, mayalso be utilized. In a competitive assay, the labeled probe is generallyconjugated with an antibody that is identical to, or an analogue of, theanalyte. Thus, the labeled antibody competes with the analyte ofinterest for the available receptive material. Competitive assays aretypically used for detection of analytes such as haptens, each haptenbeing monovalent and capable of binding only one antibody molecule.Examples of competitive immunoassay devices are described in U.S. Pat.No. 4,235,601 to Deutsch, et al., U.S. Pat. No. 4,442,204 to Liotta, andU.S. Pat. No. 5,208,535 to Buechler, et al., which are incorporatedherein in their entirety by reference thereto for all purposes. Variousother device configurations and/or assay formats are also described inU.S. Pat. No. 5,395,754 to Lambotte, et al.; U.S. Pat. No. 5,670,381 toJou, et al.; and U.S. Pat. No. 6,194,220 to Malick, et al., which areincorporated herein in their entirety by reference thereto for allpurposes.

[0050] Besides flow-through assay devices, the hollow particles of thepresent invention, as mentioned above, may also be utilized indiffraction-based assay devices (i.e., biosensors). The term“diffraction” refers to the phenomenon observed when waves areobstructed by obstacles caused by the disturbance spreading beyond thelimits of the geometrical shadow of the object. The effect is markedwhen the size of the object is of the same order as the wavelength ofthe waves. For diffraction-based assay devices, the obstacles areanalytes (with or without attached particles) and the waves are lightwaves. For example, various examples of diffraction-based assay devicesare described in U.S. Pat. No. 6,221,579 to Everhart, et al., which isincorporated herein in its entirety by reference thereto for allpurposes.

[0051] Referring to FIG. 2, for instance, one embodiment of adiffraction-based assay device 80 is shown in which a receptive material82, such as described above, is coated onto the surface of a substrate84. Any one of a wide variety of materials may serve as the substrate 84to which the receptive material 82 is applied. Such materials are wellknown to those skilled in the art. For example, the substrate 84 may beformed of any one of a number of suitable plastics, metal coatedplastics and glass, functionalized plastics and glass, silicon wafers,foils, glass, etc. Rather than requiring a rigid substrate for thephotopatterning process described herein, it has been found thatthermoplastic films are quite suitable. Such films include, but are notlimited to, polymers such as: polyethylene-terephthalate (MYLAR®),acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, cellulose triacetate, polyethylene,polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, and aromatic polysulfones. Typically, the plasticfilm has an optical transparency of greater than about 80%. Othersuitable thermoplastics and suppliers may be found, for example, inreference works such as the Modern Plastics Encyclopedia (McGraw-HillPublishing Co., New York 1923-1996).

[0052] If desired, the thermoplastic film may have a metal coating. Thefilm with metal coating thereon may have an optical transparency of fromabout 5% to about 95%. A more desired optical transparency for thethermoplastic film used in the present invention is from about 20% toabout 80%. In one embodiment, the thermoplastic film has at least about80% optical transparency, and the thickness of the metal coating is suchas to maintain an optical transparency greater than about 20%, so thatdiffraction patterns can be produced by either reflected or transmittedlight. This corresponds to a metal coating thickness of about 10 toabout 20 nanometers. However, in other embodiments, the metal thicknessmay be between approximately 1 nanometer and 1000 nanometers. Thepreferred metal for deposition on the film is gold. However, silver,aluminum, chromium, copper, iron, zirconium, platinum, titanium, andnickel, as well as oxides of these metals, may be used. Chromium oxidecan be used to make metallized layers.

[0053] The receptive material 82 may be applied to the substrate 84 byany conventional method. The receptive material 82 is applied so that itgenerally uniformly covers an entire (for example, upper) surface of thesubstrate 84. Although not required, non-contact methods for applyingthe receptive material 82 may be desired so as to eliminate thepossibility of contamination by contact during application. Suitableapplication methods include, but are not limited to, dipping, spraying,rolling, spin coating, and any other technique wherein the receptivematerial layer can be applied generally uniformly over the entire testsurface of the substrate. Simple physisorption can occur on manymaterials, such as polystyrene, glass, nylon, or other materials wellknown to those skilled in the art. One particular embodiment ofimmobilizing the receptive material layer 82 involves molecularattachment, such as that possible between thiol or disulfide-containingcompounds and gold. Typically, a gold coating of about 5 to about 2000nanometers thick is supported on a silicon wafer, glass, or polymer film(such as a MYLAR® film). The receptive material 82 attaches to the goldsurface upon exposure to a solution thereof.

[0054] The receptive material layer 82 may also be formed on thesubstrate 84 as a self-assembling monolayers of alkanethiolates,carboxylic acids, hydroxamic acids, and phosphonic acids on metallizedthermoplastic films. The self-assembling monolayers have the receptivematerial bound thereto. For instance, U.S. Pat. No. 5,922,550, which isincorporated herein in its entirety by reference thereto for allpurposes, provides a more detailed description of such self-assemblingmonolayers and methods for producing the monolayers.

[0055] Once the receptive material layer 82 is applied to the substrate84, a mask (not shown) is then placed over the substrate 84, and themask and substrate 84 combination is irradiated with an energy source88. In its basic form, the “mask” serves to shield or “protect” at leastone area or section of the receptive material 82 from the irradiatingenergy source and to expose at least one adjacent section to the energysource 88. For example, the mask may be a generally transparent ortranslucent blank (e.g., a strip of material) having any pattern ofshielded regions printed or otherwise defined thereon. The exposedunshielded regions of the mask correspond to the exposed areas of thesubstrate 84. Alternatively, the mask may simply be a single objectplaced upon the substrate 84. The area under the object would beprotected and thus define an active area of the receptive material 82,and the area around the object would be exposed to the energy source 88and thus define an area of inactive receptive material. Alternatively,the object may have any pattern of openings defined therethroughcorresponding to the exposed areas.

[0056] The mask may be formed of any suitable material that protects theunderlying portion of the substrate 84 from the irradiating energysource. A material that has proven useful for defining patterns ofactive and inactive receptive material regions on a gold-plated MYLAR®film coated with an antibody solution where the energy source is UVlight is a transparent or translucent polymer film (such as MYLAR®)having a pattern of shielded or protected regions printed thereon. Thistype of mask is useful for light sources with a wavelength equal orgreater than about 330 nanometers. For light sources having a wavelengthbelow about 330 nanometers, a quartz or fused silica mask having chromeor other metal plated shielded regions defined thereon may be used. Itmay be desired to select a hole pattern and size so as to maximize thevisible diffraction contrast between the active and inactive regions. Asone example of a pattern, it has been found suitable if the activeregions are defined as generally circular with a diameter of about 10microns and spaced from each other by about 5 microns. However, otherpatterns that provide a defined diffraction image would be suitable.

[0057] The energy source 88 is selected so that the receptive material82 exposed by the mask is rendered inactive or incapable of bindinganalyte. Without being limited by theory, it is believed that one likelymechanism is that the energy source 88 essentially destroys the bondstructure of the receptive material 82 by a radical mechanism. Theenergy source 88 is selected so that the receptive material 82 exposedby the mask is rendered inactive. The energy source 88 essentiallydestroys the bond structure of the receptive material 82 by a radicalmechanism. The receptive material 82 under the shielded areas of themask is protected during the irradiation step. Thus, upon removal of themask, a pattern of active and inactive receptive material areas aredefined. It should be understood that the term “pattern” includes as fewas one active area and one inactive area. Upon subsequent exposure ofthe diffraction-based assay device to a medium containing the analyte ofinterest, such analyte will bind to the receptive material in the activeareas. The analyte results in diffraction of transmitted and/orreflected light in a visible diffraction pattern corresponding to theactive areas.

[0058] Any suitable energy source 88 may be selected for irradiating themask and substrate combination. The energy source 88 may be, forexample, a light source, e.g., an ultraviolet (UV) light source, anelectron beam, a radiation source, etc. In one particular embodiment,the receptive material 82 is a protein-based material, such as anantibody, and the deactivating energy source 88 is a UV light source.The sensor would be exposed to the UV source for a period of timesufficient for deactivating the antibody. Wavelengths and exposure timesmay vary depending on the particular type of receptive material. Othersuitable energy sources may include tuned lasers, electron beams,various types of radiation beams including gamma and X-ray sources,various intensities and wavelengths of light including light beams ofsufficient magnitude at the microwave and below wavelengths, etc. Itshould be appreciated that any number of energy sources may bespecifically tailored for deactivating a particular antibody or othertype of biomolecule. Care should be taken that the energy source doesnot damage (e.g., melt) the underlying substrate or mask.

[0059] In some embodiments, the hollow particles of the presentinvention may be used in a diffraction-based assay device, such asdescribed above, to enhance and/or cause the desired diffractionpattern, particularly if the analyte is not of a size that results inthe desired diffraction pattern. For instance, one embodiment includesconjugating hollow particles 85 with specific binding members 87, suchas antibodies, that specifically bind to an analyte A. For detecting theanalyte A in a test sample, the test sample is first exposed to thehollow particles 85 having the specific binding members 87 thereon. Theanalyte A will bind to the specific binding members 87. Then, the hollowparticles 85 are optionally washed and exposed to the biosensor film 84with the pattern of active receptive material 82. The receptive material82 then binds to the analyte A on the hollow particles 85, therebyimmobilizing the hollow particles 85 in the same pattern as the activereceptive material 82 on the film 84. Because the bound hollow particles85 will cause diffraction of the visible light, a diffraction pattern isformed, indicating the presence of the analyte A.

[0060] Various other diffraction-based assay device configurations maybe utilized with hollow particles in accordance with the presentinvention. For instance, in one embodiment, the test sample may beapplied to the assay device simultaneously with the hollow particles.Likewise, in another embodiment, the hollow particles may be firstpre-dried on the assay device, and thereafter, the test sample isapplied thereto.

[0061] Regardless of the specific configuration of the assay device, thehollow particles of the present invention may impart a variety ofbeneficial characteristics thereto. For instance, one benefit is thatmany hollow particles, such as hollow latex polymers, form a relativelystable dispersion without the requirement of refrigeration and continualagitation. This can lessen the cost and time required for preparing theassay device and dramatically reduce particle aggregation duringconjugation with specific binding members. The particles may also bemore readily resuspended for use in the assay device. This is ofparticular advantage when the hollow particles are pre-dried onto anassay device. In addition, the hollow nature of the particles may allowthem to be more “monodispersed”, i.e., a dispersion of particles ofapproximately the same shape and/or size, than conventional latex beadsused in assay devices. This can provide improved reliability andreproducibility due to their generally uniform properties. Moreover,because the particles are hollow, they tend to have a lower weight thanmost conventional beads. The lighter weight hollow particles can movemore readily through the assay device, thereby shortening themeasurement time of the device. The hollowness of the particles alsoresults in a relatively high refraction index, which can enhance thesignal (e.g., diffraction pattern) provided by the particles in sometypes of assay devices. The refractive index may be especially enhancedin the case where the hollow particles are formed from materials with ahigh refractive index, such as titanium dioxide or gold. Thisenhancement may provide a corresponding enhancement in diffractionintensity.

[0062] The present invention may be better understood with reference tothe following examples.

EXAMPLE 1

[0063] The ability to conjugate antibodies onto hollow particles inaccordance with the present invention was demonstrated. InitiallySunSphere™ hollow particles (available from Rohm & Haas) were provided.The particles had an approximate solids content of 26% and an averagemeasured size of 300 nanometers (based on SEM and particle sizer). AnSEM photograph of such hollow particles is shown in FIG. 4.

[0064] To begin the conjugation process, 50 microliters of the hollowparticles were placed in an Eppendorf microcentrifuge tube (1.5 to 1.9milliliter capacity). A carbonate buffer was added to fill the tube andthe cap closed. The contents of the tube were centrifuged for 6 minuteson high setting (recommended=12,000 rpms). After centrifuging, thesupernatant was removed using a pipette and discarded. The resultingpellet was resuspended in a phosphate buffer by filling one half of thetube and vortexing the pellet until completely dispersed. The tube wasthen filled to capacity and centrifuged for 6 minutes at 12000 rpms. Thesupernatant was again removed and discarded, and the remaining pelletresuspended, and sonicated.

[0065] 0.75 milliliters of a 2% carbodiimide solution was then made byweighing out 15 milligrams of carbodiimide (obtained from Polysciences,Inc.) and dissolving it in 0.75 milliliters of phosphate buffer. To theredispersed pellet, 0.6 milliliters of the carbodiimide solution wasadded drop wise. Thereafter, the solution and pellet were mixed for 30minutes at room temperature on a shaker that provided end-to-end mixing.The mixed contents were centrifuged for 6 minutes, with the resultingsupernatant being discarded. The pellet was resuspended in a boratebuffer (obtained from Polysciences, Inc.) and centrifuged, with thesupernatant again being discarded. The pellet was resuspend in 1.2milliliters of the borate buffer and sonicated for 5 minutes.

[0066] 800 micrograms of a thiolated, monoclonal antibody to C-reactiveprotein (obtained from BiosPacific, Inc.) was then added to theresuspended pellet and left overnight at room temperature with gentleend-to-end mixing. Thereafter, the mixture was centrifuged for 10minutes. The supernatant was discarded or retained for proteindetermination using UV/Vis at 280 nanometers. The resulting pellet wasthen resuspended in 1 milliliter of 0.1 molar ethanolamine (obtainedfrom Polysciences, Inc.) and mixed gently for 15 minutes at roomtemperature. This step served to block the unreacted sites on themicroparticles. The suspension was centrifuged for 6 minutes, with thesupernatant being discarded. Finally, the pellet was resuspended in 1milliliter of a storage buffer to a final concentration of 1.25% solids.

EXAMPLE 2

[0067] The ability to form a diffraction-based assay device with theconjugated hollow particles of Example 1 was demonstrated.

[0068] A gold-coated plastic film obtained from CP Films, Inc. of CanogaPark, Calif. was initially provided. The film included about 10nanometers in thickness of gold on one side of a MYLAR film (thicknessof 7 mils) so that the resulting film had a resistance of less than orequal to 13 ohms/square. The film was soaked in a 5-milligram permilliliter solution of β-casein (a blocking agent). The solution of wasprepared by dissolving 25 milligrams of β-casein in 5 milliliters ofphosphate buffered saline (PBS) at a pH of 7.2. After exposure to theβ-casein solution for 10 minutes, the film was rinsed with distilledwater and dried in an air stream. The treated film, gold-side up, wasthen contact-printed with a thiolated, monoclonal antibody to C-reactiveprotein (obtained from BiosPacific, Inc.) in 10-micron diameter circleson the film to provide a patterned x-y array of the antibody on thefilm. Next, the hollow particles of Example 1 were re-suspended inphosphate buffered saline [PBS] at a pH of 7.2, which contained TritonX-100 at a concentration of 0.3%.

[0069] A test sample of CRP antigen was prepared at a concentration of50 micrograms per milliliter, and 34 microliters of the test sample wasadded to the top of the film. Simultaneously, 34 microliters of acontrol test sample was added to another piece of the contact-printedgold/mylar surface. After incubation for 5 minutes at room temperature,an 11-microliter aliquot of the re-suspended hollow particles was addedto all the samples. After incubation for 10 minutes, a nitrocellulosewicking pad (obtained from Millipore, inc. 8-micrometers pore size) wasplaced on the hollow particles, and the antigen mixed on the patternedfilm (still gold-side up on film). This caused the test sample to bewicked radially away from the gold-coated surface as it was taken in orabsorbed by the wicking pad. After the liquid sample had been absorbedby the wicking pad, a clear path for viewing diffraction (or lackthereof) remained through the hole cut from the wicking pad. The wickingpad had a 1.6-millimeter hole cut in its center using a die punch priorto placing it on the film. This small area was not applied with thewicking pad to provide a viewing area for diffraction from the sample.

[0070] The above steps provided a three-step diagnostic device.Diffraction was monitored by passing a red helium-neon laser (wavelengthof 633 nanometers) through the film. 3 orders of diffraction weredetermined to be present in the positive or CRP-spiked sample, therebyindicating a high level of analyte.

EXAMPLE3

[0071] The ability to form a diffraction-based assay device with theconjugated hollow particles of Example 1 was demonstrated.

[0072] A gold-coated plastic film obtained from CP Films, Inc. of CanogaPark, California was initially provided. The film included about 10nanometers in thickness of gold on one side of a MYLAR film (thicknessof 7 mils) so that the resulting film had a resistance of less than orequal to 13 ohms/square. The film was soaked in a 5-milligram permilliliter solution of β-casein (a blocking agent). The solution of wasprepared by dissolving 25 milligrams of β-casein in 5 milliliters ofphosphate buffered saline (PBS) at a pH of 7.2. After exposure to theβ-casein solution for 10 minutes, the film was rinsed with distilledwater and dried in an air stream. The treated film, gold-side up, wasthen contact-printed with a thiolated, monoclonal antibody to C-reactiveprotein (obtained from BiosPacific, Inc.) in 10-micron diameter circleson the film to provide a patterned x-y array of the antibody on thefilm. Next, the hollow particles of Example 1 were re-suspended inphosphate buffered saline [PBS] at a pH of 7.2, which contained 10 wt %sucrose and a heterophilic blocking reagent (obtained from Scantibodies,Inc. of Santee, Calif.) in a HBR-to-particle suspension volume/volumeratio of 1:3.

[0073] An 11-microliter aliquot of the re-suspended hollow particles wasadded to the patterned film using a pipette. The film was placed in afreezer at about −20° C. until the particle suspension was frozen(typically >1 hour), and then freeze-dried in a Labconco freeze dryingunit (with a vacuum pump, about 5 to 20 mm Hg) to dry the hollowparticles on the patterned film surface. The above provided a two-stepdiagnostic device. For testing, 34 microliters of a test sample, i.e.,3.4 microliters CRP-free whole blood with EDTA as anti-coagulant,diluted in 30.6 microliters of PBS with 0.3% Triton, was added to thehollow particles pre-dried on the film. This whole blood had either beenspiked with C-reactive protein (e.g., 50 micrograms per milliliter, forfinal concentration of 5 micrograms per milliliter after dilution) toserve as a positive sample, or left “as is” to serve as the negativecontrol. After an incubation period of 10 minutes, a nitrocellulosewicking pad (obtained from Millipore, inc. 8-micrometers pore size) wasplaced on the hollow particles, and the antigen mixed on the patternedfilm (still gold-side up on film). The wicking pad had a 1.6-millimeterhole cut in its center using a die punch prior to placing it on thefilm. This small area was not applied with the wicking pad to provide aviewing area for diffraction from the sample. After the liquid samplewas absorbed by the wicking pad, a clear path for viewing diffractionremained through the hole cut from the wicking agent.

[0074] The above steps provided a two-step diagnostic device.Diffraction was monitored by passing a red helium-neon laser (wavelengthof 633 nanometers) through the film. 3 orders of diffraction weredetermined to be present from the positive sample, thereby indicating ahigh level of analyte.

EXAMPLE 4

[0075] The ability to form a diffraction-based fluidics assay devicewith the conjugated hollow particles of Example 1 was demonstrated.

[0076] A gold-coated plastic film obtained from CP Films, Inc. of CanogaPark, Calif. was initially provided. The film included about 10nanometers in thickness of gold on one side of a MYLAR film (thicknessof 7 mils) so that the resulting film had a resistance of less than orequal to 13 ohms/square. A fresh pipette tip was used to spread out 100microliters of a thiolated, monoclonal antibody to C-reactive protein(obtained from BiosPacific, Inc.). The antibody solution was spread asevenly as possible over the gold/MYLAR surface. The antibody was allowedto incubate for 10 minutes on the film's surface. The inked film wasthen rinsed with deionized water for 10 seconds and air dried withfiltered air. The antibody-inked film was placed, gold side up, in a5-inch square vacuum chuck. Using a cheese cloth, the photo mask waswiped to remove dust and streaks. The vacuum pump was turned on toapproximately 14 pounds per square inch and the film/mask set-up wassealed with a PDMS gasket. A collimating lens (convex lens) was placedon top of the photo mask and gasket, and this entire setup was thenplaced 8.4 inches from a laser lamp source (225 nanometers) and exposedfor 2 minutes. The areas not exposed to UV light were marked and cut outfrom the film. The remaining patterned film was then cut into thedesired size for later use.

[0077] Next, the hollow particles of Example 1 were re-suspended inphosphate buffered saline [PBS] at a pH of 7.2, which contained 10 wt %sucrose and a heterophilic blocking reagent (obtained from Scantibodies,Inc. of Santee, Calif.) in a HBR-to-particle suspension volume/volumeratio of 1:3. The particles were striped onto the patterned film using aKinematic module Matrix 1600™ (set to a platen speed of 7 centimetersper second) to achieve a dispense rate of 0.9 microliters percentimeter. The pump was initially purged, which allows the particlesuspension to flow through the tubing to the nozzle head. The stripingdistance was adjusted to meet the length of the film, and lined up toensure that striping was done down the center of the film.

[0078] After full-length striping, the film was placed immediately intoa freezer at −20° C. to freeze the particle suspension, e.g., about 1hour or more. The film was then freeze-dried for 18 hours while on flatice packs (˜5 to 20 mm Hg, using Labconco Model # 77500 freeze dryingunit with a vacuum pump). The sample was then removed and placed in alow humidity chamber (e.g. ≦30% relative humidity).

[0079] A fluidics assay device was then formed from the above-mentionedhollow-particle printed film. The fluidics assay was cast from etchedsilicon masters or photoresist-made masters with polydimethylsiloxane(PDMS) (Sylgard 184 from Dow Chemical, Co.). The fluidic channelscontained a 400-micron inlet channel with 92 wicks at 50 microns each.The hollow particle-printed film was removed from the freeze-dryer andcut to fit the PDMS fluidic assay, i.e., 2 centimeters×2 centimeters.The PDMS fluidic assay was mounted on the film in a manner that alignedthe hollow particles along the fluidics inlet channel and centraldetection zone.

[0080] A 1-microliter aliquot of diluted blood (e.g., 1 part whole bloodwith EDTA as anti-coagulant in 9 parts diluent containing phosphatebuffer solution (PBS) at pH 7.2 with Triton X-100 at 0.3%) was thenplaced at the entrance of the inlet channel. The whole blood was eitherleft “as is” to serve as a negative control, or spiked with 10 to 50micrograms per milliliter C-reactive protein (e.g., corresponding to 1to 5 micrograms per milliliter CRP after dilution). The blood aliquotwas allowed to flow up to the central detection zone area via capillaryaction, and finally through the wicks of the fluidic assay.Incubation/wicking times, i.e., the time for fluid to enter the inletchannel and then evacuate the central detection zone, generally rangedfrom 30 seconds to 8 minutes. Diffraction was monitored by passing a redhelium-neon laser (wavelength of 633 nanometers) through the film. 3 ormore orders of diffraction were determined to be present from thepositive sample, thereby indicating a high level of analyte.

EXAMPLE 5

[0081] The ability to conjugate and label hollow particles in accordancewith the present invention was demonstrated. Initially, 500 microlitersof SunSphere™ hollow particles (available from Rohm & Haas) were washedtwice with 1 millimeter of 2-(N-morpholino) ethane sulfonic acid (pH of5.3). Thereafter, 30 milligrams of carbodiimmide was added to the hollowparticle/buffer solution. The compounds were allowed to react for 10minutes under constant rotation.

[0082] The hollow particles were separated from the reaction solutionand washed with 1 millimeter of a borated buffer. 1 milligram offluorescent dye(5-(and-6)-((N-(5-aminopentyl)amino)carbonyl)tetramethylrhodamine(tetramethylrhodamine cadaverine) was added to 1 millimeter of thehollow particle/borate buffer solution. The compounds were allowed toreact for 1 hour under constant rotation. After the reaction wascomplete, the supernatant was discarded and the hollow particles werewashed with borate buffer until the supernatant solution became clear.The hollow particles were then re-suspended into 1 millimeter of boratebuffer. From this solution, 100 microliters were removed and diluted in500 microliters of borate buffer. 100 microliters of monoclonal antibodyMab5811 (BiosPacific, Inc., 6.4 milligrams per milliliter) was added tothe hollow particle solution and allowed to react under constantrotation for over 56 hours. The reaction was quenched with 200microliters of ethanolamine, and the hollow particles were then washedwith a PBS buffer and stored in 500 milliliters of a storage buffer thatcontained 0.1 molar PBS, 0.15 molar NaCl ,1% BSA, 5% glycerol and 0.1%NaN₃.

EXAMPLE 6

[0083] The ability to form a lateral flow assay device with the hollowparticles of Example 5 was demonstrated.

[0084] A nitrocellulose porous membrane (HF 120 from Millipore, Inc.)having a length of approximately 30 centimeters was laminated ontosupporting cards. CelQuat™ 100-H (a cellulosic polyelectrolyticderivative available from National Starch & Chemical, Inc.) was strippedonto the membrane to form a control line. In addition, monoclonalantibody for C-reactive protein (Mab 5804 obtained from BiosPacific,Inc., concentration of 1 milligram per milliliter) was immobilized onthe porous membrane to form a detection line. The membrane samples werethen dried for 1 hour at a temperature of 37° C. A cellulosic fiberwicking pad (Millipore, Inc. Co.) was attached to one end of themembrane and cut into 4-millimeter half strips. The half stick stripswere put into various micro-wells containing 20 microliters of thefluorescent hollow particles of Example 5 and 20 microliters of CRPantigen solutions or 20 microliters of TBS buffer. The sample containingonly TBS buffer was used as a negative control, while the samplescontaining the CRP antigen in TBS were used as test samples.

[0085] Upon completion of the assay, the half stick was removed from themicro-well and the fluorescent intensity was then measured on thedetection line using a Fluorolog III Spectrofluoremeter (SPEXIndustries, Inc., Edison, N.J.) with a right angle mode. The results areshown below in Table I, wherein “I” represents the measured signalintensity from the fluorescent hollow particles. TABLE I SignalIntensity Results Analyte (nanograms per milliliter) Signal Intensity“I” 0 44 (control) 5 115 50 160 500 240

[0086] The signal intensity for the control was considered backgroundand did not represent any significant analyte concentration. Inpractice, this background intensity would be subtracted from allmeasured results.

[0087] While the invention has been described in detail with respect tothe specific embodiments thereof, it will be appreciated that thoseskilled in the art, upon attaining an understanding of the foregoing,may readily conceive of alterations to, variations of, and equivalentsto these embodiments. Accordingly, the scope of the present inventionshould be assessed as that of the appended claims and any equivalentsthereto.

What is claimed is:
 1. An assay device comprising a plurality ofdetectable probes, wherein at least one of said detectable probescontains a particle that defines a hollow interior constituting fromabout 20% to about 100% of the spatial volume occupied by said particle.2. An assay device as defined in claim 1, wherein said hollow interiorconstitutes from about 30% to about 100% of the spatial volume occupiedby said particle.
 3. An assay device as defined in claim 1, wherein theaverage size of said particle ranges from about 0.1 nanometers to about1,000 microns.
 4. An assay device as defined in claim 1, wherein theaverage size of said particle ranges from about 0.1 nanometers to about100 microns.
 5. An assay device as defined in claim 1, wherein theaverage size of said particle ranges from about 1 nanometer to about 10microns.
 6. An assay device as defined in claim 1, wherein said particlehas a spherical shape.
 7. An assay device as defined in claim 1, whereinsaid particle is formed from a polymer.
 8. An assay device as defined inclaim 7, wherein said particle is formed from a core polymer and a shellpolymer.
 9. An assay device as defined in claim 1, wherein said particleis formed by electrostatic layer deposition.
 10. An assay device asdefined in claim 1, wherein said at least on detectable probe furthercontains a label.
 11. An assay device as defined in claim 1, whereinsaid particle is conjugated with a specific binding member.
 12. An assaydevice as defined in claim 1, wherein the assay device is a flow-throughassay device.
 13. An assay device as defined in claim 1, wherein theassay device is a diffraction-based assay device.
 14. A flow-throughassay device for detecting an analyte in a test sample, saidflow-through assay device comprising a detection zone immobilized with areceptive material, said detection zone being in fluid communicationwith detection probes containing particles that individually define ahollow interior constituting from about 20% to about 100% of the spatialvolume occupied by said particle, wherein said detection probes arecapable of generating a detection signal while within said detectionzone so that the amount of the analyte within the test sample isdetermined from said detection signal.
 15. A flow-through assay deviceas defined in claim 14, wherein said hollow interior constitutes fromabout 30% to about 100% of the spatial volume occupied by said particle.16. A flow-through assay device as defined in claim 14, wherein saidparticles are conjugated with a specific binding member for the analyte.17. A flow-through assay device as defined in claim 14, wherein saiddetection probes contain a label selected from the group consisting ofchromogens, catalysts, fluorescent compounds, chemiluminescentcompounds, phosphorescent compounds, radioactive compounds, directvisual labels, liposomes, and combinations thereof.
 18. Adiffraction-based assay device for detecting an analyte of interest in atest sample, said assay device comprising: a substrate having areceptive material applied to at least one surface thereof, saidreceptive material being specific for the analyte; detection probescontaining particles that individually define a hollow interiorconstituting from about 20% to about 100% of the spatial volume occupiedby said particle; and wherein upon exposing said substrate to the testsample, the analyte is capable of binding with said receptive materialon said substrate to result in a detectable diffraction pattern uponsubsequent exposure of said substrate to a light source.
 19. Adiffraction-based assay device as defined in claim 18, wherein saidhollow interior constitutes from about 30% to about 100% of the spatialvolume occupied by said particle.
 20. A diffraction-based assay deviceas defined in claim 18, wherein said substrate is selected from thegroup consisting of plastics, metal coated plastics and glass,functionalized plastics and glass, silicon wafers, metal oxides, glass,foils, and combinations thereof.
 21. A diffraction-based assay device asdefined in claim 18, wherein said substrate comprises a polymer filmcoated with a metal.
 22. A diffraction-based assay device as defined inclaim 21, wherein said metal is selected from the group consisting ofgold, silver, chromium, nickel, platinum, aluminum, iron, copper,titanium, zirconium, corresponding oxides of these metals, andcombinations thereof.
 23. A diffraction-based assay device as defined inclaim 18, wherein said receptive material is printed in a pattern onsaid substrate, and wherein a blocking material is further applied tonon-printed areas of said substrate.
 24. A detectable probe for use inan assay device, wherein the detectable probe contains a particle thatdefines a hollow interior constituting from about 20% to about 100% ofthe spatial volume occupied by said particle, said particle beingconjugated with a specific binding member.
 25. A detectable probe asdefined in claim 24, wherein said hollow interior constitutes from about30% to about 100% of the spatial volume occupied by said particle.
 26. Adetectable probe as defined in claim 24, wherein the average size ofsaid particle ranges from about 0.1 nanometers to about 1,000 microns.27. A detectable probe as defined in claim 24, wherein the average sizeof said particle ranges from about 0.1 nanometers to about 100 microns.28. A detectable probe as defined in claim 24, wherein the average sizeof said particle ranges from about 1 nanometer to about 10 microns. 29.A detectable probe as defined in claim 24, wherein said particle has aspherical shape.
 30. A detectable probe as defined in claim 24, whereinsaid particle is formed from a polymer.
 31. A detectable probe asdefined in claim 30, wherein said particle is formed from a core polymerand a shell polymer.
 32. A detectable probe as defined in claim 24,wherein said particle is formed by electrostatic layer deposition.
 33. Adetectable probe as defined in claim 24, wherein said specific bindingmember is covalently bonded to said particle.